Project Summary/Abstract - Project 2: Lung cancer is the second most prevalent and most lethal cancer in the United States [1]. Despite recent advances, 5-year survival has remained essentially unchanged for the last 40 years at 11-17% [2] and additional approaches are urgently needed to improve outcomes. Pharmacological ascorbate (P-AscH-; high dose intravenous vitamin C) has recently re-emerged as an agent that enhances cancer cell responses to radiation and chemotherapy in cell culture and in animal models. Preliminary data show selective toxicity as well as chemo-radiosensitization of human non-small cell lung cancer (NSCLC) versus normal non- transformed bronchial epithelial cells (HBEpC) with P-AscH- treatment. However the mechanisms underlying the differential susceptibility of lung cancer vs. normal cells to P-AscH- are not known. Based on strong pre- clinical and clinical data from an ongoing clinical trial in NSCLC, Project 2 will test the hypothesis that P-AscH- selectively sensitizes NSCLC cells to radiation and chemotherapy by increasing cancer cell steady- state levels of H2O2 as a result of specific disruptions in redox-active iron metabolism mediated by endogenous levels of O2?-/H2O2. This hypothesis will be tested mechanistically in preclinical models in Aim 1 as well as in a phase 1B/2 clinical trial in stage III inoperable lung cancer patients in Aim 2. Aim 1 will determine in vitro and in vivo if differential regulation of redox-active labile iron pools by O2?- and H2O2 causes alterations in Fe metabolism (i.e. transferrin receptor, ferritin, Fe-S proteins) that mediate P-AscH--induced radio-chemotherapy sensitization in NSCLC vs. normal lung epithelial cells. Aim 2 will determine in a phase 1b/2 clinical trial if combining P-AscH- with radiation + Carbo/Taxol can increase treatment efficacy in stage IIIA/B inoperable NSCLC subjects as determined by increases in median overall survival. Biomarkers of FDG uptake pre- and post-treatment as determined by FDG PET-CT imaging, transferrin saturation, 4HNE-modified proteins, and circulating levels of labile Fe will be determined in the clinical trial and correlated to clinical responses. The successful completion of this project will define biochemical mechanisms involving O2?-/H2O2 mediated disruptions in iron metabolism underlying P-AscH--mediated selective toxicity and radio-chemo- sensitization in NSCLC vs. normal cells as well as providing a new paradigm for using P-AscH- clinically to exploit fundamental differences in cancer vs. normal cell metabolism for increasing treatment efficacy in stage IIIA/B inoperable lung cancer subjects using traditional radio-chemotherapies.